The tricarboxylic acid (TCA) cycle is of central importance in cellular metabolism, yet there has not been a detailed investigation of the regulation of any gene encoding a TCA cycle enzyme, in either a prokaryotic or eukaryotic cell. The mechanism of regulation of genes encoding citrate synthase in the eukaryotic microorganism Saccharomyces cerevisiae (baker's yeast) will be defined. Citrate synthase catalyzes the first and the rate- limiting step of the TCA cycle within the mitochondria. The TCA cycle is required for the utilization of nonfermentable carbon sources (in the absence of glucose). The first three TCA cycle enzymes, citrate synthase, aconitase, and isocitrate dehydrogenase, are also required for the synthesis of alpha-ketoglutarate, the precursor for glutamate. Yeast contains two nuclear genes encoding functional citrate synthase: CIT1 (major, mitochondrial isozyme) and CIT2 (minor, nonmitochondrial isozyme). Transcription of both genes is reduced synergistically by glucose and glutamate in the growth medium, resulting in cellular economy. To define DNA sequences required for this novel pattern of regulation, we have begun constructing deletions of DNA upstream of either gene. To simplify assaying gene expression we have fused CIT1 and CIT2 to the lacZ gene (beta-galactosidase) of E. coli. DNA sequences located more than 150 base pairs upstream of the transcription initiation regions of CIT1 and CIT2 are necessary and sufficient (in either orientation) for gene activation and regulation by glucose and glutamate. Further deletion mapping and oligonucleotide-based methods will be used to precisely define these DNA sequences. Mutants defective in trans-acting factors (e.g., DNA-binding proteins) required for regulation of citrate synthase genes will be using CIT1 or CIT2 lacZ fusions and the chromogenic substrate Xgal. Catabolite derepressed expression of CIT1 (but not CIT2) requires the HAP2,3,4 DNA- binding complex, which also activates various cytochrome genes. This is the first evidence for a mechanism coordinating regulation of the TCA cycle and the electron transport chain, at the level of gene expression. We will also determine whether any complexes formed in vitro between yeast proteins and upstream DNA sequences which regulate CIT1 or CIT2 in vivo are dependent on the HAP2,3,4 loci or other loci defined by regulatory mutations (see above). Our long term goal is to understand how requirements for the catabolic and biosynthetic functions of various portions of the mitochondrial TCA cycle are communicated to nuclear genes encoding TCA cycle enzymes.